The studies detailed in this proposal seek to understand myosin-1 interactions with membranes and membrane components at the molecular level and to elucidate the role of these interactions in membrane organization and dynamics. Myosin-1 family members (eight different genes expressed in humans) are single-headed motor proteins that play essential roles in membrane dynamics and other cellular processes. A key property of myosin-1 isoforms appears to be their ability to directly link the actin cytoskeleton to cell membranes. Several myosin-1 isoforms have been shown to bind directly to membranes, and one isoform, myo1c, has been shown to bind tightly to phosphoinositide headgroups via a putative pleckstrin homology domain in its tail domain. Given the importance on the myosin-1 family in driving membrane dynamics, it is remarkable that the membrane interactions of most of the myosin-1 have been unexplored. Therefore, this proposal is focused on characterizing the myosin-1-membrane interactions. We will use a variety of biochemical and biophysical techniques to examine membrane binding specificities, affinities, and dynamics. The specific aims are as follows: Aim 1: Binding of myosin-1 family members to membranes and phosphoinositides Subaim 1.1: Characterization of phosphoinositide binding Subaim 1.2: Phosphoinositide specificity Subaim 1.3: Kinetics of binding Aim 2: Interplay between membranes and the actomyosin system Subaim 2.1 Reconstitution of actin motility on membrane surfaces Subaim 2.2 Myosin-mediated PIP2 clustering Subaim 2.3 Dynamics of myosin-1 binding to PIP2 in a membrane. PUBLIC HEALTH RELEVANCE: Myosin-1 family members are important for numerous basic cellular processes. In humans, myosin-1 mutations have been linked to deafness, while myosin-1 deletions in mice result in increased susceptibility to infection, kidney inflammation, and defects in the gut endothelium. Understanding the specificity of and roles for myosin-1 interactions with lipid components of the cell membrane will provide insight into these normal and diseased physiological states.